Purification and characterization of mouse α-lactalbumin from lactating mammary glands

The whey protein, α-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14 00). Antibodies prepared against these peptides precipitate newly synthesized and secreted α-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse α-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine α-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of α-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect α-lactalbumin levels as low as 0.25 ng.     

Galactosyl transferase-the liver plasma membrane binding-site for asialo-glycoproteins

Agalacto-fetuin inhibits the binding of ¹²⁵I-asialo-fetuin by liver plasma membrane fragments. The chemically prepared agalacto-glycoprotein derivative is not a substrate for plasma membrane sialyl transferase and therefore this indicates that agalacto-fetuin is a true inhibitor of the membrane binding of ¹²⁵I-asialo-fetuin. The plasma membrane fraction also contains galactosyl transferase activity and the binding of ¹²⁵I-asialo-fetuin by plasma membranes is prevented by α-lactalbumin, a known inhibitor of glycoprotein-galactosyl transferase. These data indicate that galactosyl transferase is the liver plasma membrane component which binds asialo-glycoproteins.     

The calcium-dependent electrophoretic shift of alpha-lactalbumin, the modifier protein of galactosyl transferase

alpha-Lactalbumin, the modifier protein of galactosyl transferase in the synthesis of lactose by the mammary gland, has been shown to undergo a Ca2+-dependent electrophoretic shift. Such shifts, characteristic of most calcium modulated proteins, are related to gross conformational changes upon binding calcium when detected in the presence of detergent (SDS-PAGE). However, we detected the calcium shift for alpha-lactalbumin using non-denaturing PAGE (ND-PAGE) where electrical charge changes are observed upon binding calcium. In order for a shift to be observed between the apo and calcium bound protein, calcium ion binding to proteins must have minimal dissociation constants (Kdiss) of 10(-7) M; alpha-lactalbumin is reported to bind calcium at Kdiss = 10(-10) to 10(-12) M. The electrophoretic shift identifies alpha-lactalbumin in complex milk whey patterns of many species of mammals.     

A sensitive and reproducible procedure for the assay of arginyl-tRNA: Protein arginyl transferase

The activity of arginyl-tRNA: protein arginyl transferase was found to be enhanced four- to sevenfold by substituting bovine α-lactalbumin for bovine serum albumin, the standard acceptor protein used thus far. With α-lactalbumin as the acceptor protein in place of serum albumin, a sensitive and reproducible procedure for the transferase assay was established.     

Casein and alpha-lactalbumin messenger RNAs during the development of mouse mammary gland. Isolation, partial purification, and translation in a cell-free system

Messenger RNAs for the milk proteins, casein and α-lactalbumin, were isolated and partially purified from lactating mouse mammary glands by oligo(dT)cellulose chromatography followed by sucrose density gradient centrifugation. The translation of poly(A)⁺ mRNA in a wheat germ cell-free system yielded three casein polypeptides and a putative precursor form of α-lactalbumin which were precipitated by specific antibodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The casein polypeptides synthesized in vitro had a molecular weight that was no greater than that of the caseins in mouse milk. The presence of individual casein mRNAs coding for these polypeptides was demonstrated by the translation of various fractions of mRNA obtained by sucrose density gradient centrifugation of poly(A)⁺ mRNA. Casein mRNA activity increased about 250-fold between midpregnancy and the 10th–12th days of lactation, amounting to 50–60% of the total mRNA activity in that tissue. A similar study of α-lactalbumin mRNA showed an increase during lactation amounting to 0.2–0.4% of the total mRNA activity, which corresponds to the percentage of α-lactalbumin in total mouse milk protein.     

Isolation and characterization of Golgi membranes from bovine liver

Zonal centrifugation has been used to isolate a fraction from bovine liver which appears to be derived from the Golgi apparatus. Morphologically, the fraction consists mainly of sacs and tubular elements. Spherical inclusions, probably lipoproteins, are occasionally seen in negative stains of this material. The preparation is biochemically unique. UDP-galactose:N-acetyl glucosamine, galactosyl transferase activity is concentrated about 40-fold in this fraction compared to the homogenate. Rotenone- or antimycin-insensitive DPNH- or TPNH- cytochrome c reductase activities are 60–80% of the level of activities found in microsomes. Purified organelles from bovine liver such as plasma membranes, rough microsomes, mitochondria and nuclei have negligible levels of galactosyl transferase. Some activity is present in smooth microsomes but at a level compatible with the possible presence of Golgi membranes in this fraction. The Golgi fraction does not contain appreciable amounts of enzymes such as ATPase, 5'-nucleotidase, glycosidase, glucose-6-phosphatase, acid phosphatase, or succinate-cytochrome c reductase. Similar fractions isolated from bovine epididymis also have very high levels of galactosyl transferase. The fraction is heavily osmicated when incubated for long periods of time at elevated temperatures, a characteristic property of Golgi membranes.     

α-Lactalbumin, Engineered to be Nonnative and Inactive, Kills Tumor Cells when in Complex with Oleic Acid: A New Biological Function Resulting from Partial Unfolding

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded α-lactalbumin on the stationary phase in situ. Because native α-lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human α-lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA^all-Ala), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA^all-Ala and oleic acid (rHLA^all-Ala-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA^all-Ala protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA^all-Ala-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify α-lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.     

Biosynthesis of keratan sulfate: purification and properties of a galactosyltransferase from bovine cornea.

A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl-d-glucosamine, α- and β-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl-d-glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl-d-glucosamine; the same glycosidic linkage was found in [¹⁴C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower K_m with keratan than with N-acetyl-d-glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + α-lactalbumin = lactose synthase), including modulations of acceptor specificity by α-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-β-galactopyranosyl-(1 → 4)-N-acetylglucosamine] of cornea keratan sulfate.     

Binding of naphthalene dyes to the N and A conformers of bovine α-lactalbumin

Contrary to earlier findings, monomeric native α-lactalbumin does bind naphthalene dyes such as ANS and TNS with marked enhancement of their fluorescence. Nanosecond decay measurements indicate there to be two dye binding sites per protein molecule with lifetimes of ca. 2 and 15 ns for ANS and 5 and 11 ns for TNS. The fluorescence titrations curves of α-lactalbumin with ANS and TNS reflect this site multiplicity, i.e., it was not possible to analyze such curves with a single K_diss. The apparent dissociation constants for binding of ANS and TNS to native bovine α-lactalbumin, as determined by an ultracentrifugal technique, ca. 950 and 900 μm, respectively, indicate that such binding is considerably weaker than previously supposed. The A conformer (metal ion-free form) of α-lactalbumin binds ANS and TNS more tightly than the N (native) form of the protein with marked fluorescence enhancement. The A conformer has two dye binding sites with lifetimes for ANS and TNS comparable with those seen with native protein.     

Synthesis of sialyllactosamine clusters using carbosilane as core scaffolds by means of chemical and enzymatic approaches

An efficient synthesis of sialyllactosamine (SiaLacNAc) clusters using carbosilanes as core scaffolds has been accomplished by means of chemical and enzymatic approaches. N-Acetyl-d-glucosamine (GlcNAc) clusters having O-glycosidic linkage or S-glycosidic linkage were chemically synthesized from known intermediates in high yields. The GlcNAc clusters were first used as substrates for β1,4 galactosyl transferase using UDP-galactose (UDP-Gal) as a sugar source to provide corresponding N-acetyllactosamine clusters. Further sugar elongation of the LacNAc clusters was demonstrated using α2,3 sialyl transferase and CMP-neuraminic acid (CMP-NANA) to yield the corresponding SiaLacNAc clusters.     

Cascade control of E. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade.

Enzymes and regulatory proteins involved in the cascade control of glutamine synthetase activity of Escherichia coli have been separated from one another and the effects of numerous metabolites on each step in the cascade have been determined. The adenylyl transferase (ATase) -catalyzed adenylylation of glutamine synthetase, which requires the presence of the unmodified form of the regulatory protein PII is enhanced by glutamine and is inhibited by either α-ketoglutarate (α-KG) or the uridylylated form (PII·UMP) of the regulatory protein. PII·UMP and α-KG act synergistically to inhibit this activity. In contrast, the PII·UMP-dependent, ATase-catalyzed deadenylylation of glutamine synthetase requires α-KG and ATP and is inhibited by glutamine or PII and synergistically by glutamine plus PII. The capacity of uridylyl transferase (UTase) to catalyze the uridylylation of PII is dependent on the presence of α-KG and ATP and is inhibited by glutamine. The deuridylylation of PII·UMP by the uridylyl removing enzyme (UR) is enhanced by glutamine but is unaffected by α-KG. However, CMP, UMP, and CoA all inhibit activity at 10^?6m. High concentrations of ATase inhibit both UR and UTase activities, presumably by binding the regulatory protein. Of more than 50 substances that alter the activity of at least one enzyme in the cascade, only α-KG and glutamine affect the activity at every step. This accounts for the observation that glutamine synthetase activity in vivo is very sensitive to the intracellular ratio of α-KG to glutamine.     

The lactose synthase acceptor site: a structural map derived from acceptor studies

Summary A pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either or -, but - is somewhat preferred. In the absence of -lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH₂) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of -lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (-configuraation). However, upon -lactalbumin binding the aglycone specificity allows for axial (-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that -lactalbumin binds to a hydrophobic site some distance from the C-2 group.     

Thermodynamics of α-lactalbumin unfolding

Thermodynamic investigations of α-lactalbumin have been performed by isothermal calorimetric guanidine hydrochloride titrations as well as by scanning calorimetric measurements in the presence and absence of guanidine hydrochloride. Compared with lysozyme, α-lactalbumin is less stable, and its changes of enthalpy and heat capacity at unfolding are lower. Thermal unfolding of α-lactalbumin can be described to the first approximation by the two-state transition model even in the presence of guanidine hydrochloride.     

GLYCOSYL TRANSFERASE ACTIVITY IN NORMAL AND SCRAPIE-AFFECTED MOUSE BRAIN

—The conditions required for the optimal activity of several glycosyl transferases were determined in normal and scrapie-affected mouse brain. Particulate preparations were analysed for fucosyl, galactosyl, mannosyl and N-acetyl glucosaminyl transferase activity using endogenous acceptors. Solubilized preparations were analysed for fucosyl, galactosyl and sialyl transferase activity using defined exogenous acceptors. The activity of each of these enzymes was followed at intervals throughout the development of scrapie in the mouse. In the endogenous system no change was found in the fucosyl and mannosyl transferase activities of the scrapie material, but the levels of galactosyl and N-acetyl glucosaminyl transferase began to rise as clinical signs of scrapie developed i.e. at 15–16 weeks post-inoculation. In the exogenous system the levels of galactosyl and fucosyl transferase began to rise in the scrapie material at 11–12 weeks post-inoculation, rising to twice the normal value at 18–20 weeks. Sialyl transferase showed no change in activity.     

Conformational stability of α-lactalbumin missing a peptide bond between Asp66 and Pro67

The peptide bond between Asp⁶⁶-Pro67 of α-lactalbumin was cleaved with formic acid (cleavedα-lactalbumin). Secondary structural changes of the cleavedα-lactalbumin, in which the two separated polypeptides were joined by disulfide bridges, were examined in solutions of sodium dodecyl sulfate (SDS), urea, and guanidine hydrochloride. The structural changes of the cleavedα-lactalbumin were compared with those of the intact protein. The relative proportions of secondary structures were determined by curve fitting of the circular dichroism spectrum. The cleavedα-lactalbumin contained 29%α-helical structure as against 34% for the intact protein. Some helices of the cleavedα-lactalbumin which had been disrupted by the cleavage appeared to be reformed upon the addition of SDS of very low concentration (0.5mM). In the SDS solution, the helicities of both the intact and cleaved proteins increased, attaining 44% at 4mM SDS. On the other hand, the helical structures of the cleavedα-lactalbumin began to be disrupted at low concentrations of guanidine hydrochloride and urea compared with that of the intact protein. However, no diffrence was observed in the thermal denaturations of the intact and cleaved proteins, except for the difference in the original helicities. The helicities of both proteins decreased with an increase of temperature up to 65°C and recovered upon cooling.     

Further evidence for an active site polypeptide of galactosyl transferase

In a previous report it was shown that galactosyl transferase activity after blotting from acrylamide gel was present in a molecular weight range of less than 14 kDa, in Triton X-100 (1). Molecular sieve chromatography on Superose 12, in the presence of Triton X-100, gave the same result. The low molecular weight activity peak was eluted together with peptides as a part of the covalent structure of the enzyme or as absolutely requires effectors. Peptide mapping showed a new poly-lysine-like peptide and a new hydrophobic peptide in this low molecular weight activity peak as effectors of the enzyme inside its hydrophobic environment.     

Temporal effect of prolactin on the activities of lactose synthetase, alpha-lactalbumin, and galactosyl transferase in mouse mammary gland explants

The onset of the prolactin (PRL) stimulation of lactose synthesis is between 4 and 8 hr after adding PRL to cultured mouse mammary tissues. The synthesis of lactose is catalyzed by the enzyme lactose synthetase, which is composed of two parts, alpha-lactalbumin and galactosyl transferase. In time-sequence studies, it was found that the activity of galactosyl transferase is enhanced by PRL in concert with the onset of the PRL stimulation of lactose synthesis. In contrast, the earliest detectable effect of PRL on alpha-lactalbumin activity occurred 24 hr after adding PRL to the cultures. It is, therefore, apparent that the rate-limiting component for the PRL stimulation of lactose synthesis in cultured mouse mammary tissues is galactosyl transferase activity.     

Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

α-Lactalbumin and dimyristoyl phosphatidylcholine were used as a prototype to study the influence of a protein conformational change, induced by the pH, on the interaction between that protein and a phospholipid.The enthalpy changes associated with the interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles were measured as a function of the molar ratio of phospholipid to protein, pH and temperature. Gel-filtration, electron-microscopic and fluorescence data for the same experimental conditions were also obtained. At pH 4 and 5, the enthalphy changes (ΔH) are not only larger than at physiological pH, but also show a maximum at about 23°C in the ΔH vs. temperature graph. At pH 6 and 7, on the contrary, ΔH increases with decreasing temperature without a maximum in the curve. Gel-chromatographic and electron-microscopic data show that at pH 6 and 7, the morphological characteristics of the vesicles are unchanged upon addition of α-lactalbumin, while at pH 4 and 5 at 23°C an extra peak appears in the gel-filtration graphs between the pure vesicles and α-lactalbumin. The new fraction contains lipid-protein complexes. Electron micrographs show that bar-shaped entities are formed. A red shift at 23°C and a blue shift at 37°C, both to 336 nm, are observed for λ_max of the fluorescence emission spectra at pH 4 when α-lactalbumin is brought into contact with the phospholipid. At the same time, a strong increase in the fluorescence intensity is observed. The chromatographic and fluorescence data indicate that a lipid-protein complex with a molar ratio of approx. 80 is formed. At pH 7 and different temperatures, the emission maximum remains at the wavelength of pure α-lactalbumin, the change in the fluorescence intensity, however, indicates that interaction with the lipid occurs.The results can be explained on the basis of an electrostatic interaction at pH 6 and 7, and a hydrophobic interaction at pH 4 and 5.     

Subcellular fractionation of Trypanosoma brucei bloodstream forms with special reference to hydrolases

Bloodstream forms of Trypanosoma brucei have been screened for the presence of enzymes that could serve as markers for the plasma membrane, flagellar pocket, lysosomes, endoplasmic reticulum and Golgi apparatus in order to study the subcellular organization of the digestive system of the parasite. Acetylesterase, acid DNase, acid phosphatase, acid phosphodiesterase, acid proteinase, acid RNase, alanine aminotransferase, galactosyl transferase, alpha-glucosidase, inosine diphosphatase and alpha-mannosidase were partially characterized and their assays optimized for pH-dependent activity, linearity of reaction with respect to incubation time and enzyme concentration, and the effect of inhibitors and activators. The association of these enzymes with particulate material and the presence of structural latency were investigated. Acid proteinase and alpha-mannosidase are particle-bound and latent in cytoplasmic extracts; they can be activated and solubilized in part by Triton X-100. Similar results were obtained for acid phosphatase, acid phosphodiesterase and inosine diphosphatase. Neutral alpha-glucosidase, though partly sedimentable, does not show latency and is readily solubilized by the detergent. Galactosyl transferase is firmly membrane-bound even in the presence of 0.1% Triton X-100. Cell fractionation by differential centrifugation and density equilibration on sucrose gradients revealed that both alpha-mannosidase and acid proteinase are associated with organelles that band at a density of about 1.20 g/cm3. Inosine diphosphatase, galactosyl transferase, acid phosphatase and acid phosphodiesterase sediment predominantly as microsomal constituents equilibrating at densities between 1.13 and 1.15 g/cm3. In addition, inosine diphosphatase and galactosyl transferase exhibit considerable activity at higher densities (1.18-1.25 g/cm3). Neutral alpha-glucosidase is mainly recovered in the nuclear and microsomal fraction; its particulate part equilibrates as a single band at rho = 1.22 g/cm3. Acetylesterase and acid DNase are largely soluble, whereas acid RNase does not produce distinct sedimentation and banding profiles. In intact cells, neutral alpha-glucosidase and acid phosphatase appear to be highly accessible to their substrates. It is tentatively concluded that (a) acid proteinase and alpha-mannosidase are lysosomal enzymes, (b) acid phosphatase and acid phosphodiesterase are associated with the flagellar pocket and part of the former enzyme probably with the endoplasmic reticulum, (c) galactosyl transferase is a constituent of the Golgi apparatus, and (d) alpha-glucosidase may serve as a marker for the plasma membrane. Inosine diphosphatase may also be derived from the latter structure.     

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: Effect of charge,fluidity and antigen-to-lipid ratio

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens α-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6′-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic α-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that α-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions.